A facile method for the construction of oligonucleotide microarrays.
In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.
Sethi
Dalip
D
Institute of Genomics and Integrative Biology (CSIR), Delhi University Campus, Delhi, India.
Kumar
A
A
Gupta
K C
KC
Kumar
P
P
D020137:Base Pair Mismatch; D001483:Base Sequence; D005898:Glass / Q000737:chemistry; D007700:Kinetics; D016920:Meningitis, Bacterial / Q000175:diagnosis / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011437:Propylamines / Q000737:chemistry; D015203:Reproducibility of Results; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties; D013696:Temperature
C487481:N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine; D009838:Oligodeoxyribonucleotides; D011437:Propylamines; D012821:Silanes; C082343:silanol; C069686:gamma-glycidoxypropyltriethoxysilane